We have very poor viability after freezing of pure antibiotic-selected murine iPSC-derived cardiomyocytes in a medium composed of 10%DMSO+90%FBS. Any suggestions on how to improve the freezing protocol to increase the viability of cells after thawing.
Hello Tomo,
don't have experience with the following but it sounds good:
"Cryopreservation of hESC-derived cardiomyocytes
Differentiated cultures containing beating cardiomyocytes were dissociated with 0.25% trypsin/0.5 mmol/l ethylenediaminetetraacetic acid (Invitrogen) and neutralized with defined trypsin inhibitor (Cascade Biologics). The cells were washed and resuspended by slowly adding cryopreservation solution CryoStor™ CS-10 (BioLife Solutions Inc.). The final cell concentrations were approximately 5–10 × 106 cells in 0.25 ml/vial (small scale) or 4–8 × 107 cells in 1.5 ml/vial (large scale). Cells were frozen using a controlled rate freezer at −1°C/min before the temperature reached −40°C and −5°C/min from −40°C to −80°C. Vials were then transferred to a liquid nitrogen tank after reaching −80°C. To thaw the cells, the vials were incubated in a water bath at 37°C until no ice crystals were visible. The cell suspension was transferred to a 15 or 50 ml tube and then slowly diluted with RPMI/B27. After centrifugation at 450 g for 5 min, the supernatant was removed and cells were resuspended in RPMI/B27. The cells were subjected to flow cytometric analysis, cultured on Matrigel (BD Biosciences)-coated plates in RPMI/B27 or prepared for transplantation as described below. Comparison of cell recoveries from samples containing cardiomyocytes of different purities (indicated as mean ± standard deviation of the percentage of cardiac troponin I [cTnI], α-actinin or Nkx2.5) was carried out by Tukey’s multiple comparison test using Prism software."
This is from that paper:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3057133/pdf/nihms269347.pdf
The cells are generated in a special way but I think this make no difference for freezing and thawing.
Maybe you wanna try this method if no body here can give a better protocol.
Best from Münster
Sven
Dear all,
at present I cannot suggest any other useful protocol. But, I hope to find the opportunity/fundings to perform some more experiments with a method (Irdani et al., 2011) so far successfully applayed to worms and hopefully suitable for some other biological samples.
Sincerely yours
Tiziana
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